The mitochondrial electron transport chain (ETC) is central to the survival of all eukaryotic cells, driving the synthesis of ATP that fuels cellular bioenergetics. Despite its fundamental role, significant variation exists in the structure of the ETC in different organisms. Unlike the relatively simple ETC characteristic of higher animals, plants possess a complex, branched respiratory chain containing type II NAD(P)H dehydrogenases (ND) and alternative oxidases (AOX), which provide alternative pathways for electron flow. Several recent studies have suggested that these alternative respiratory enzymes may minimize electron leakage from the ETC, diminishing the production of damaging reactive oxygen species (ROS). ROS production and resulting oxidative stress are of significant biomedical interest, since oxidative damage appears to play a significant role in aging as well as a diverse array of pathologies, from Alzheimer's to diabetes. Notably, the progressive oxidative damage associated with aging in animals is absent in plants, and the expression of an ND in an animal system (Drosophila) decreases mitochondrial ROS production and increases lifespan. Thus, the overarching goal of this proposal is to define the relationship between ETC structure and ROS production/progressive oxidative damage. Toward this end, we plan to experimentally modify the plant ETC by using an inducible RNA interference vector to silence the AOX gene family, the NDinternal gene family, and the NDexternal gene family in the model plant species Arabidopsis thaliana. The resulting transgenic plants (independent AOX-silenced, NDin-silenced, and NDout-silenced lines) will allow the regulated suppression of distinct alternative respiratory pathways, creating intermediates between plant-type and mammalian-type respiratory chain configurations. To link these unique respiratory structures to quantifiable effects on cell physiology, we plan to measure ROS production, oxidative damage, and the size and oxidation state of cellular antioxidant pools in the transgenic lines. In addition, we will examine global changes in the transcriptomes of the transgenic lines in order to characterize how altered respiratory chain structure affects ROS-associated signaling pathways. This proposal expands and builds upon current NIH SCORE-funded research focused on the development of Arabidopsis as a model system to study basic cellular redox biology and oxidative damage. Overall, the proposed project will have a major impact on our fundamental understanding of mitochondrial- associated ROS production, a process which is central to both the basic field of cell biology and the maintenance of human health.